Infectious salmon anaemia (ISA) is a virus disease limited to Atlantic salmon (Salmo salar L.) in cultivation. The disease was diagnosed first time in 1985 in parr (the Bremnes outbreak). The disease has only been found in Atlantic salmon in salt water or salt water added to fresh water. Administrational measures issued by the authorities, as stamping out (and isolation) of affected fish farms followed by disinfection and restrictions on trading/moving of fish in the area have limited the number of outbreaks from the peak around 1990, but in the later years, the disease has increased in extension. Primarily, ISA has been a Norwegian salmon production problem, and in 1998 15 outbreaks were registered. However, during the period 1997–98, ISA was found and verified in Canada (97) and Scotland (98).
The disease is caused by a general infection which among others cause severe anaemia and bleeding lesions. The disease spreads slowly in an infected fish farm, and the mortality can vary from 15 100%. There is no available cure against ISA. The goal of implemented control measures is to minimalise the risk for exposure of ISA virus to the salmon. The demonstration of infection implies stamping out of all fish in an affected fish farm and disinfection of the localities. There is no available vaccine.
Due to the severe economical strains the disease implies to society in general and to the individual fish farmer, a good and secure diagnostic procedure is of importance. Diagnosis of ISA is still based on a combination of macroscopical and microscopical observations of dead/dying fish (pathological/histological investigations). Recently, researchers have had success in growing ISA virus in cell culture (1), which is very time- and resource demanding. An indirect immune fluorescence test for the demonstration of infective material has been developed for the use in tissue sections and tissue impressions (2). A quick-test, —a RT-PCR (reverse transcriptase polymerase chain reaction)—test to demonstrate ISA virus in salmon has been developed as well. It can also be used in ISA infected fish showing no clinical signs of disease (3). This test is ready for the use in the context of diagnosis and mass investigation.
The most likely preventive action against ISA is the development of a vaccine and other influences of the natural defence system in the salmon.
ISA virus contains a negatively charged single-stranded RNA genome of 8 segments. The total size of the segments is 14,5 Kb (1,5×103 base pairs). The virus replicates itself in the nucleus. It is a 100–120 nm enveloped virus with 10 nm peplomers, and it separates itself from the cell membrane by budding. The entrance of ISA virus in cells is pH-dependant. ISA virus harbours hemagglutinating and hemadsorbent abilities (3). All listed features indicate that the virus belongs to the family Orthomyxoviridae, implying an influenza-like virus.
The immune system of salmon share many similar properties with the immune system of mammals. Accordingly, it is possible to draw a number of parallels. Teleosts posses immunocompetent cells such as B- and T-lymphocytes, lymphokines, complement factors and they produce immunoglobulines. Farmed salmon is vaccinated against important bacterial infections. In Norway, vaccines against the IPN virus is also available, but the effect of these vaccines is discussed. The demand for new and more effective vaccines against virus diseases in farmed fish is substantial. DNA-vaccine is an important candidate among vaccine strategies to choose and has been described in various contexts (4). At DNA immunisation against for instance flue virus, protective effects not only directed against the actual antigen variant of the virus used in the vaccine, but also effect against antigenically different virus have been observed (5). This broad immune response can possibly be explained by a good cellular response. It is assumable that a good cellular immune response provides a far better protection against ISA than humoral immune response alone. This is due to the fact that the cellular immune response is directed against a broader range of antigens, and the cellular response is longer-lasting than the humoral response.
The interferon system is also an interesting part of the teleost immune system against influenza-like viruses including ISAV. The interferons induce retardation of virus replication and are of particular importance before the establishment of a specific immune response. Interferon-induced proteins, known as Mx-proteins, are important in the retardation process of influenza-like viruses. For instance, mice lacking functional Mx-genes do not survive influenza infections (6). Mx-genes are also demonstrated in salmon (7), but these do not seem to repress ISA virus sufficiently to prevent disease. The ISA virus has possibly adopted to salmon to such a degree that it may replicate despite the Mx-response of the host. Mx-proteins from human as well as mouse appear to restrict the replication of ISA virus in cell cultures (8).
There is a relatively large degree of homology between Mx-genes from mammals, birds and fish, indicating the severe threat of influenza-like viruses to the species, creating a selection pressure to the benefit of individuals carrying Mx-genes. It is therefore assumable that influenza-like viruses have existed in the marine environment over a substantial period of time. Farming of salmonids in sea-water has established conditions for an effective cultivation and distribution of virus, disease outbreaks represent reminders of the existence of influenza-like viruses in the marine environment. A virus reservoir in the marine environment has not yet been identified, thus complicating preventive measures.
Electron microscope studies have demonstrated that ISA virus buds from endothelial cells in blood vessels in several different organs (9). Following experimental challenge tests, virus particles have been identified in most organs, making the disease different from influenza infections in humans where infection usually is limited to the respiratory system. Orthomyxovirus possess 3–4 different surface proteins,; hemagglutinin is regarded to be of particular importance, being responsible for choice of host cell, this due to receptor recognition and thus the binding to the host cell. Hemagglutinin is likely to harbour similar abilities in the ISA virus. Host-cell restricted and surface located protein splitting enzymes (proteases) are necessary to activate the hemagglutinin, making the transport of virus into the cell possible. In this context, the accessibility and tissue distribution of suitable proteases in addition to the accessibility of cellular surface molecules which can act as receptors for ISA virus is of importance. The wide-spread tissue distribution of ISA virus during infection indicates that if the infectibility is dependant on proteolytic activation of virus proteins, this activation is conducted by ordinarily existing proteases. This can partly explain the pathogenicity of the ISA virus which may give up to 100% mortality in certain outbreaks.
Previously, procedures for immunisation of aquatic species by DNA expression systems have been described. See European Patent application no. 839913/964713 (NO). Herein is described the procedure of immunisation using DNA vaccines directed against various aquatic viruses, ISA virus is not described but mentioned in the Norwegian application in claim 11 page 39. Any specific references concerning ISA virus are not mentioned, neither with respect to which gene sequences which may be efficient, nor methods to sequence them.
The difference between human and fish vaccine is limited. Presently, no DNA vaccine is available commercially. The principle is the same, but the application will be different, and of course infective agent. A limited number of vaccines against virus diseases are available in aquaculture production. DNA vaccines represent a new and promising approach in this context. DNA-vaccination implicates administration of antigen-expression vectors which give protein synthesis in situ in tissues in the vaccinated animal. DNA vaccines have experimentally been shown to give protection against influenza virus in mice (close relative to ISA virus) (10, 11, 12, 13). In contrast to recombinant or subunit vaccines, DNA vaccines will mimic attenuated or living, recombinant vaccines due to their possibility to initiate the production of cytotoxic T-cell responses and antibody responses which recognise authentic conformation dependant epitopes. The matrix proteins in orthomyxovirus is by number the predominant protein in the virus particle and has been demonstrated to be of importance to give cross protection (e.g. protection against different strains of influenza-virus which would give reduced protection due to antigen/genetic drift if this was not the case) in mice (14). The matrix protein should therefore be a part of a DNA-vaccine which should protect against ISA virus (5,10).
The traditional fish vaccines are injected intraperitoneally, and an admixture of adjuvance to increase the effect is used. Oil mixtures based on animals/vegetables are mainly used, which may cause severe side effects in the context of peritonitis which may lead to fusions and reduced appetite. DNA vaccines do not demand adjuvance of this kind to be effective. In certain cases, the use of liposomes may increase the effect, but a good response following intracutaneous and intramuscular injections without admixtures is expected. It is also planned to investigate if sufficient effect after dip- or bath vaccination is raised.